Supplemental Figure S1
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Fig. S1. Western blot analysis, with the anti-V-H+-PPase antibody, of cellular fractions separated by differential centrifugation into P13 and P100 insoluble and S100 soluble fractions. Proteins from the supernatant were precipitated with trichloroacetic acid. All fractions were resuspended in 20 µl of SDS-PAGE loading buffer for Western blot analysis. To determine if one of the two proteins recognised by the V-H+-PPase antibody could be specific to acidocalcisomes, we performed cell fractionations and differential centrifugations to see if the two proteins were associated to different cellular compartments. 5×108 promastigotes were osmotically and mechanically lysed (in 1 ml of hypotonic buffer: 1 mM Tris-HCl pH 8.0, 1 mM EDTA, containing 1× complete protease inhibitor cocktail (Roche) mix and 10 µM E-64, and they were subsequently disrupted by passing them several times through a 26-gauge needle). Unbroken cells were removed by low speed centrifugation at 300g. The cell-free extract was then subjected to sequential centrifugations at 13,000 × g and at 100,000 × g. The low speed pellet (P13) is thought to contain membrane-associated proteins from the plasma membrane, endoplasmic reticulum (ER), or mitochondria, whereas the P100 fraction is expected to be enriched for Golgi and endosomes or other vesicular membranes. Cell fractionation experiments showed that the two proteins were insoluble, hence probably membrane-associated, but segregated to different membrane fractions. The high molecular weight protein was mostly found in the P100 fraction, where vesicular organelles such as acidocalcisomes could be expected. Whereas the lower molecular weight protein, still present in the Δap3δ cell extracts, was predominantly in the P13 fraction. These data tend to confirm that the high molecular weight protein corresponding to the V-H+-PPase and absent from the Δap3δ mutant is indeed acidocalcisome-specific. However, given the heterogeneity in composition of acidocalcisomes, it cannot be excluded that the remaining minor protein could correspond to a protein still present on the membrane of a population of acidocalcisomes with different properties.
